Phosphorus compound from animal proteids and process of manufacture thereof



Patented Jan. 7,, 1930 UNITED STATES PATENT OFFICE.

BWIGEL POSTERNAK Ann 'rnnononn r'os'rnRNAx, or cnnnn-nouemms, 1cm

GENEVA, SWITZERLAND, Assmnons 'ro .THE rum SOCIETY or cnnmrcAn mnus'ray m BASLE, or BASEL, SWITZERLAND rnosrnomrs courounn FROM ANIMAL PROTEIDS Ann rnocnss or mAmAc'rum THEREOF No Drawing. Application filed March 21. 1927, Serial No. 177,222, and in Switzerland March 81 1928.

The present invention relates tofnew phosphorus compounds from the proteids of egg yolk, said compounds being useful in thera- Y peut-ics, for instanceas tonics, and it comprises the new compounds. themselves as well as the process of their manufacture.

In the U. S. Patent No. 1,690,752 a process is described according to which, by subjecting 'egg yolk in succession to a peptic and tryptic digestion, mixtures of thephosphorus compounds of the proteids contained in egg yolk are obtained which may be isolated in the free form or as salts. T The present invention relates to a process for separatingthese phosphorus compounds,

' which have been distinguished in the said specification by the letters a, B and from each other and obtaining each in the free stateor in form of salts.

The phosphorus compound a is only slight ly soluble in cold water and completely insoluble in solutions of inorganic salts made acid with mineral acids,.by which salts it is therefore easily precipitated. It forms soluble alkali salts and insoluble alkali earth salts. It contains phosphorus and nitrogen Physio 24, page 173 [1906]; Levene and Alsberg, Journ. Biolo Chem. vol. 2, page 217 [1906]). The our phosphonc acid groups are apparently esterified with the hydroxyl groups of the 3 serines and of the acid containm 6 carbon atoms. The phosphorus content 0 the compound moves between 12.5 and 13.5 per cent.

The phos 'horus com ound B isthe' iron carrier of. t e egg yo itsphysical and chemical properties are similar to those of the phosphorus compound a. It contains age 505 about 3.5 per cent of iron, and therefore more than 7 times as much as the preparation of Hugounenq and Morel contains (Comptes rendus 140, page 1065 [1905]) and 12 times as much as the Haematogen of Bunge contains (Ztschr. f. Physiol. Chemie vol. 9, page 49 [1885]). The phosphorus content amounts to about 12 per cent and the atomic ratio of phosphorus to nitrogen is 1 :2. v

The phosphorus compound 7 is fundamentally different from a and B compounds. It is very soluble in Water, gives soluble alkali earth salts and insoluble heavy metal salts. Its phosphorus content amounts to about 8 per cent. The atomic ratio of phosphorus to'nitrogen is 1:3; It gives the Biuret and Molischreactions but not the Millons reacl t1on.

The phosphorus compound a amounts to about per cent, the phosphorus compound/ B to about 27 per cent, and the phosphorus compound 7 to about 18 per cent of the total phosphorus of the proteids of egg-yolk.-

The process accordingto this invention is 5 I based upon the followin observations (1) When alcohol is a ded, while stirring, to an aqueous solution of a mixture of the sodium salts of the three phosphorus compounds in egg yolk, which solution may bemade by dissolving the's'odium salts made as described n U. S. Patent No. 1,690,752, there is iformed suddenly 'a flocculent precipitate which contains the compound B containing 1I0I1.

(2)'- From a mixture of the sodium salts of p the phosphorus compound on and phosphorus nucleus y soluble alkali earth salts precipitate the corresponding salt of the phosphorus.

compound a which is insoluble in Water. The

phosphorus compound is not precipitated in this manner, since it yields alkali earth salts soluble in water.

(3) When an aqueous solution of a mixture of the sodium" salts of the phos horus com-- pounds a, B and. y is saturated with a neutral salt, for instance common salt,- and mineral acid is added, the phosphorus compounds 1:

and B are precipitated while the phosphorus compound y remains in solution.

(4 The phosphorus compound can he precipitated from its aqueous solution in the form of free acid or in the forin of its alkali earth salt by addition of alcohol.

Example 1 1.0 kilos of egg yolk previously extracted with boiling alcohol, dried and finely ground, are suspended in 500 litres of water and there are added 2.8 kilos of concentrated hydrochloric acid and 100 grams of pepsin or of a corresponding quantity of ydrochloric acid extract of the mucous membrane of the stomach of a ig; the mixture is heated with constant stirrin to 37-40" C. until the egg yolk has passe into solution. After about 10 hours 3.8 kilos of crystallized sodium acetate or 1 kilos of crystallized sodium carbonate and l'kilo of lacial acetic acid are added. After filtration, preferably in a filter press, the solid matter is washed with water and taken up with a solution of 1200 rams of anhydrous sodium carbonate in 40 itres of water, 100 grams of finely minced pancreas are added and the mixture is dige'sted for 24-48 hours at 40 C. It is then slightly acidified with acetic acid, filtered from undissolved matter and mixed with lead acetate'solution so long as a precipitate is produced. The lead precipitate is filtered, washed with water and stirred with a sodium carbonate solution of 20 er cent stren h until the mixture shows a eeble red on p enolphthalein paper. The phosphorus compounds ass into solution in the form of sodium sa ts while the lead remains undissolved in the form of carbonate. The solution of the sodium salts is strongl concentrated in a vacuum, and then mixe with alcohol to sl 'oduce a precipitate which is subsequently ried in a vacuum. 4

The mixture of the sodium salts of the three compounds containing phosphorous thus obtained is dissolved in 10 litres of water.

Tothis solution 2 to 3 litres of'alcohol are added graduall There is formec suddenly a flocculent precipitate," whichis filtered, re-dissolved in insolub pound ,9 is a water, if necessary with addition of some sodium carbonate, again preci itated by adding alcohol, washed and drie in a vacuum. The sodium salt of the. phos horous comellowishpow or, somewhat ta-rdily soluble in water.

The solution of this salt may be converted by double decomposition with soluble alkali earth salts, or heavy metal salts, into the corresponding alkali earth salts or metal salts of-the phosphorous compound ,8 which are e in water, for instance the magnesium salt, calcium salt, iron salt or mercu xiy salt. 7 he alcoholic filtrate from the hos horous compound p is freed from alcoho by istillatlon in a vacuum and mixed with a solution ofcalcium chloride or calcium acetate, wherewith continuous stirring.

by the calcium salt of phosphorous com ound a is precipitated. The precipitate is fi tered, washed with water and dried. The calcium salt thus obtained is a white powder insoluble in water and tasteless. B treatment with the calculated proportion o the sodium oxalate white hot, the calcium salt can be converted into the correspondingsodium salt, from which by double decomposition with a soluble alkali earth salt or heavy metal salt various alkali earth and heavy metal salts respectively may be made.

Finally, the filtrate from the calcium salt of the phosphorous com ound a is either precipitated b means of a cohol in order to obtam the ca cium salt in solid form or is precipitated by means of lead acetate and the recipitate decomposed with sulphuretted hy rogen; the lead sul hide is filtered, the excess of sulphuretted hy rogen expelled fromthe filtrate by a current 0 air'and the solution precipitated with alcohol, the precipitate being washed with alcohol and dried in a vacuum. The white powder thus obtained is the phosphorous compound 7 in free condition.-

From this powder, soluble in water b neutralization with an alkali, an alkali eart salt or a heavy metal salt, the correspondin salts can be obtained. The'sodium salt 0 the phos horous com ound can be obtained directly y treating t e aforesaid precipitate roduced by lead acetate with'sodium caronate solution.

It is obvious that for precipitating the phosphorous compound 7 another heavy metal salt may be substituted for the lead salt, for instancea salt of copper or mercury, and that for obtaining the alkali salt from the heavy metal salt another alkali compound may be used instead of soda, for instance potash.

Example 2 The mixture of sodium salts of the three phosphorous com ounds obtained as described in Exam e 1 from 10 kilos of egg yolk extracted w1th boiling alcohol, is .dls-

solved in 10 litres of water, the solution is chloric acid. This precipitate is a mixture of the phosphorous compound a and phos-f phorous compound ,3. It is dissolved in a fee 7 suitable quantity of sodium carbonate solution and mixed, with constant stirring, with such a quantity of alcohol that a sudden precipitation occurs. This precipitate is the phosphorous compound )3 and is further worked ugas described in Exam le 1. The alcoholic l trate containin the p 0s horous compound a is either free from alco 01 and mixed with soluble alkali earth salt for recipitating the phosphorus compound as i earth salt or is poured into alcohphacidified with hydrochloric acid to precipitate the phosphorus compound in the form of free,

acid.

It is obvious that the ambit of this invention is not limited to the methods of separation specified in the examples, but includes any equivalent separation method, being based on the different solubilities of the three phosphorus compounds, be it in their free form, be it in form of their salts, 1n solvents, such as for instance water or alcohol.

\Vhat we claim is 1. A process for separating at least one of the three phosphorus compounds of the proteids contained in egg yol by preparing an aqueous solution of a mixture of the alkali salts of the three phosphorus compounds, and precipitating from said solution by means of alcohol the alkali salt of the phosphorus compound B.

2. A process for separating at least one of the three phosphorus com ounds of the proteids contained in egg yol by preparing an aqueous solution of a mixture of the alkali salts of the three phosphorus compounds, precipitating from said solution by means of alcohol'the alkali salts of the phosphorus compound B, filtering and treating the filtrate containing the mixture of the alkali salts of the phosphorus compounds or and y with a soluble alkali earth metal salt, thus precipitating the alkali earth metal salt of the phosphorus compound a. 3. A process for separating at least one of the three phosphorus com ounds of the proteids contained in egg yol by preparing an aqueous solution of a mixture of the alkali salts of the three phosphorus compounds, precipitating from said solution by means of alcohol the alkali salt of the phosphorus compound B, filtering, treating the filtrate containing the mixture of the alkali salts of the phosphorus compounds or and y with'a soluble alkali earth metal salt, whereby the alkali earth metal salt of the phosphorus compound a is precipitated, then filtering and isolating from the filtrate the phosphorus compound 'y. '4. A process for separating at least one of the three phosphorus com ounds of the proteids contained in egg yol by preparing an aqueous solution of a mixture of the alkali salts of the three phosphorus compounds, precipitating from said solution by means of alcohol the alkali salt of the phosphorus compound B, filtering, treating the filtrate containing'the mixture of the alkali salts of the phosphorus compounds on and y with a soluble alkali earth metal salt, whereby the alkali earth metal salt of the phosphorus compound or is precipitated, then filtering and precipitating from the filtrate the phosphorus compound y in form of its alkali earth metal salt by means of alcohol.

66 5. A process for separating at least one of salts of the three phosphorus compounds, precipitating from said solution by means of alcohol the alkali salt of the phosphorus compound B, filtering, treating the filtrate containing the mixture of the alkali salts of the phosphorus compounds on and y with a soluble alkali earth metal salt, whereby the alkali earth metal salt of the phosphorus compound a is precipitated, then filtering and precipitating the filtrate with a heavy metal salt thus isolating the corresponding heavy metal salt of the phorphorus compound y.

6. The phosphorus compound at, which constitutes the main part of the mixture of the phosphorus compounds at, B and 1/, obtained from the proteids of egg yolk, and forms a white powder, containlng between 12.5 and 13.5 per cent of phosphorus, being but slightly soluble in cold water and insoluble in solutions of inorganic salts made acid with mineral acids, forming soluble alkali salts and insoluble alkali earth and heavy metal salts, giving the Biuret reaction but no Millon and Molisch reaction, and being useful for therapeutic purposes, for instance as a tonic.

7. The phosphorus compound B, obtained from the proteids of egg yolk, containing about 3.5 per cent of iron and about 12 per cent of phosphorus, forming alkali salts somewhat tardily soluble in Water, and alkali earth and heavy metal salts difiicultly soluble or insoluble in water, and being useful for therapeutic purposes, for instance as a tonic.

8. he phosphorus compound obtained from the proteids' of egg yolk, containing about 8 per cent of phosphorus, being very easily soluble in water, giving soluble alkali and alkali earth metal salts, and insoluble heavy metal salts; yielding the Biuret and Molisch reaction but notthe Millons reaction, and being useful for therapeutic purposes, for instance as a tonic.

In witness whereof we have hereunto signed our names this 7th day of March 1927. SWIGEL POSTERNAK.

THEODORE PQSTERNAK. A, 

